ObjectiveTo detect the function of I BHV-1 TK-/gE-/ EGFP+ gene-deleted mutant virus strain, find a new method to prevent infectious bovine rhinotracheitis (IBR), the charaction of TK-/gE-/ EGFP+ gene-deleted mutant virus strain was further studied. Methods Southern hybridization, western dot blot and Plaques test were employed in this investment. ResultsTK gene is deleted and the mutant cannot produce glycoprotein E when gE gene is deleted. The diameter of mutant strain much smaller than that of field strain, the mutant strain grows on MDBK cells and the virus titer can keep 106.0TCID₅₀/0.1 mL for more than 40 hours, which indicated that the mutant strain inherit instantly. Conclusion The gene-deleted mutant virus strain was successful constructed in this study, and the mutant virus strain was stable. This will be useful for the further study of IBR.